The goal of the proposed studies in Project 5 is to test the hypothesis that cytochrome P450-catalyzed formation of epoxyeicosatrienoic acids (EETs) in the renal cortex is involved in the renal autoregulatory physiologic responses to acute and chronic volume and/or pressure overload. We suggest that two sites of actions for this regulatory role for EETs are 1) the preglomerular arteriolar tone; and 2) proximal tubular reabsorptive rates. Since chronic increases in glomerular pressure and volume, or chronic "mismatch" between clearance demands and available filtration surface, are associated with the development of programmed nephron destruction, the induction of renal cortical arachidonate epoxidation and the role of EETs in regulating renal vascular and tubular functions in response to these stimuli, may constitute important modulatory influences in the natural course and ultimate outcome of "hyperfiltration"-associated progressive renal insufficiency. To investigate this central hypothesis, three specific aims will be addressed; in the first specific aim, we will use a combination of in vivo and in vitro models to investigate the roles of cP450 epoxide derivatives of arachidonic acid (EETs) in renal autoregulatory responses to acute and chronic increases in renal perfusion pressure, intravascular volume expansion, and glomerular hyperfiltration. these studies will examine the effects of EETs in the regulation of afferent arteriolar vasoconstriction and proximal tubular reabsorption. To address the physiology and pathophysiology of renal autoregulatory function, the in vivo models to be used are salt feeding in normal rats, Dahl hypertensive rats and extensive ablation of renal mass. Using in vitro cell systems of cultured mesangial cells, we will investigate mechanisms of cellular regulation of these compounds, including the mechanisms of these compounds in regulating contractility, transcellular metabolism by cyclooxygenase and determination of whether specific membrane binding sites are present. In vitro studies in the proximal tubule will be directed toward understanding the mechanisms by which these compounds increase calcium influx, and the role of these compounds in regulation of net sodium reabsorption. The second specific aim of these studies is to elucidate the signaling mechanisms mediating alterations in both acute (increased production) and chronic (enzymatic induction) activity of epoxygenase and omega/omega-1 hydroxylase. In vivo and in vitro studies will address the potential roles of intrarenal physical forces, hormonal systems (Atrial Natriuretic Peptide, Endothelial Derived Relaxing Factor, Renin-Angiotensin-Aldosterone System) and renal nerves in the regulation of cP450 arachidonate metabolite production. The in vivo systems utilized will include salt loading, Dahl hypertension and renal ablation, as mentioned above. The in vitro models will examine alterations in activity and enzyme levels in renal microvascular endothelial cells, mesangial cells and proximal tubule cells subjected to hormonal stimulation, mechanical stretch or alterations in extracellular milieu. These combined in vivo and in vitro studies will address mechanisms of production and secretion of these metabolites, the role of posttranslational regulation of enzyme activity and the role of transcriptional control in the regulation of enzyme levels. A third aim of Project 5 is to investigate the biologic actions of omega/omega-1 metabolites of arachidonic acid in the normal rat kidney and renal cells in culture, concentrating specifically upon their potential regulatory functions in the glomerular microvasculature and the proximal tubule. In summary, the proposed combination of in vivo and in vitro methodologies will allow intensive investigation of the roles of the cP450 arachidonate metabolites in the regulation of renal autoregulatory responses, and it is hoped that these proposed studies will increase our understanding of both mechanisms by which the kidney normally responds to alterations in perfusion pressure and volume expansion, as well as dysregulation seen in various pathophysiologic conditions.